Clonal deletion in the thymus of potentially autoreactive T cells remains the major mechanism of maintaining tolerance in the central lymphoid system. Since, under certain circumstances (autoimmunity, extrathymic T cell development and transplantation), the first T cell engagement with antigen occurs in the periphery, distinct mechanisms must be either inherent or introduced therapeutically to prevent T cell reactivity. The major goals of this proposal are: to study the T cell response to antigens expressed in the peripheral tissue; to manipulate the T cell/antigen-presenting cell interaction to promote T cell tolerance; to determine the underlying basis for T cell unresponsiveness in vivo; and to use gene therapy and transgenic mouse technology to manipulate T cell responses in vivo. In order to accomplish these goals, we will: 1) Study the role of co-stimulatory molecules in the induction of T cell tolerance following transplantation. These studies will use a number of unique reagents that regulate co-stimulatory molecules including: mAbs (anti- murine CTLA-4, anti-CD28, anti-B7 and anti-ICAM-1); CTLA4lg; CD28- deficient and CTLA-4-deficient mice; and B7/Islet beta cell and CTLA4lg/keratinocyte transgenic mice. 2) To develop novel gene therapy approaches to introduce inhibitors of co-stimulation directly into transplanted tissue as a means of blocking transplant recognition. In these studies, adenovirus-medicated gene transfer will be used to introduce the genes for co-stimulation antagonists, such as CTLA4lg, directly into the pancreatic islets. In addition, a transgenic mouse expressing CTLA4lg in keratinocytes will be used in transplantation experiments to study the effect of local immunosuppression on proximally and distally transplanted tissue. 3) To study peripheral T cell tolerance of intraepithelial lymphocytes (IELs) in T cell receptor transgenic mice exposed to nominal antigen expressed on "non- professional" antigen presenting cells. Although potentially autoreactive T cells are effectively deleted from the lymphoid tissue, T cells that develop extrathymically, such as IELs, are present, express normal levels of the TCR, but are functionally inactivated. However, following continuous antigen exposure, these cells undergo ligand-induced cell death (apoptosis) not unlike what is observed in the thymus. The present studies will examine the nature of the biochemical signalling defect(s) in these cells and determine whether the induced unresponsiveness is a result of similar genetic changes [i.e., induction of cell death genes [bcl-x] or transcription factors] as has been observed in vitro. In addition, we will use the CD28-deficient and CTLA4lg transgenic mice to determine the role of CD28/B7 in this peripheral tolerance model. Finally, we will use the adenovirus to introduce co-stimulatory molecules in an attempt to reverse tolerance as a model for autoimmune inflammatory bowel disease. Together these studies will provide insights into the basis of peripheral T cell tolerance in vivo and provide novel therapeutic approaches to tolerance induction.